Access to the Antenna System of Photosystem I via Single-Molecule Excitation-Emission Spectroscopy.

In the development of single-molecule spectroscopy, the simultaneous detection of the excitation and emission spectra has been limited. The fluorescence excitation spectrum based on background-free signals is compatible with the fluorescence-emission-based detection of single molecules and can provide insight into the variations in the input energy of the different terminal emitters. Here, we implement single-molecule excitation-emission spectroscopy (SMEES) for photosystem I (PSI) via a cryogenic optical microscope. To this end, we extended our line-focus-based excitation-spectral microscope system to the cryogenic temperature-compatible version. PSI is one of the two photosystems embedded in the thylakoid membrane in oxygen-free photosynthetic organisms. PSI plays an essential role in electron transfer in the photosynthesis reaction. PSIs of many organisms contain a few red-shifted chlorophylls (Chls) with much lower excitation energies than ordinary antenna Chls. The fluorescence emission spectrum originates primarily from the red-shifted Chls, whereas the excitation spectrum is sensitive to the antenna Chls that are upstream of red-shifted Chls. Using SMEES, we obtained the inclining two-dimensional excitation-emission matrix (2D-EEM) of PSI particles isolated from a cyanobacterium, Thermosynechococcus vestitus (equivalent to elongatus), at about 80 K. Interestingly, by decomposing the inclining 2D-EEMs within time course observation, we found prominent variations in the excitation spectra of the red-shifted Chl pools with different emission wavelengths, strongly indicating the variable excitation energy transfer (EET) pathway from the antenna to the terminal emitting pools. SMEES helps us to directly gain information about the antenna system, which is fundamental to depicting the EET within pigment-protein complexes.

Development of Line-Detected UV-Vis Absorption Microscope and Its Application to Quantitative Evaluation of Lithium Surface Reactivity.

Electrochemical reactions in practical batteries occur in confined environments where anode and cathode electrodes are separated only by a thin separator. Therefore, their electrochemical behaviors may differ from those obtained in the conventional experimental cells, where the two electrodes (working and counter electrodes) are largely separated compared to the batteries. The spatial and temporal distributions of the chemical species in the vicinity of each electrode are highly expected to be determined for quantitatively understanding the phenomena in confined environments. In the present study, we developed a line-detected UV-vis absorption microscope that simultaneously measures space-resolved UV-vis absorption spectra. This novel technique has been successfully applied to evaluate the reactivities of the highly reactive lithium (Li) surfaces in organic electrolyte solutions under in situ conditions. The quantitative evaluations of the dissolution rate of Li and the diffusion constant of the product were successfully realized by analyzing the space- and time-resolved absorption spectra based on Fick's law of diffusion. The microscopic technique is expected to open the door to understanding the fundamental electrochemistry in batteries.

Uphill energy transfer mechanism for photosynthesis in an Antarctic alga.

Prasiola crispa, an aerial green alga, forms layered colonies under the severe terrestrial conditions of Antarctica. Since only far-red light is available at a deep layer of the colony, P. crispa has evolved a molecular system for photosystem II (PSII) excitation using far-red light with uphill energy transfer. However, the molecular basis underlying this system remains elusive. Here, we purified a light-harvesting chlorophyll (Chl)-binding protein complex from P. crispa (Pc-frLHC) that excites PSII with far-red light and revealed its ring-shaped structure with undecameric 11-fold symmetry at 3.13 Šresolution. The primary structure suggests that Pc-frLHC evolved from LHCI rather than LHCII. The circular arrangement of the Pc-frLHC subunits is unique among eukaryote LHCs and forms unprecedented Chl pentamers at every subunit‒subunit interface near the excitation energy exit sites. The Chl pentamers probably contribute to far-red light absorption. Pc-frLHC's unique Chl arrangement likely promotes PSII excitation with entropy-driven uphill excitation energy transfer.

Accumulation of quenched LHCII around PSI in Chlamydomonas cells in state2 revealed by cryo-fluorescence lifetime imaging.

Fluorescence-spectral microscope observations of photosynthetic organisms at cryogenic temperatures have the ability to spectrally resolve the two photosystems (PSs) and thus provide a powerful tool to elucidate the functional analysis of photosynthesis in vivo. In the present study, a measurement channel of the fluorescence lifetime at 680 nm was added to the cryo-microscope system previously developed by the authors. This provides access to information on the functional state of the light-harvesting system in living cells during regulation by a mechanism called state transitions. The observations of state1-locked and state2-locked Chlamydomonas cells at 80 K enabled us to identify a component showing rapidly decaying fluorescence with a lifetime of ca. 3 ps and emitting at around 676 nm. The component was assigned to the light-harvesting complex II (LHCII) that is isolated from both PSs and in a quenched state, probably due to the formation of aggregates. Simultaneous spectral observations revealed the accumulation of this free LHCII in the photosystem I (PSI)-enriched region within each state2-locked cell. To the best of our knowledge, this is the first in-vivo observation which suggests the localization of the quenched LHCII aggregates.

State transition is quiet around pyrenoid and LHCII phosphorylation is not essential for thylakoid deformation in Chlamydomonas 137c.

Photosynthetic organisms have developed a regulation mechanism called state transition (ST) to rapidly adjust the excitation balance between the two photosystems by light-harvesting complex II (LHCII) movement. Though many researchers have assumed coupling of the dynamic transformations of the thylakoid membrane with ST, evidence of that remains elusive. To clarify the above-mentioned coupling in a model organism Chlamydomonas, here we used two advanced microscope techniques, the excitation-spectral microscope (ESM) developed recently by us and the superresolution imaging based on structured-illumination microscopy (SIM). The ESM observation revealed ST-dependent spectral changes upon repeated ST inductions. Surprisingly, it clarified a less significant ST occurrence in the region surrounding the pyrenoid, which is a subcellular compartment specialized for the carbon-fixation reaction, than that in the other domains. Further, we found a species dependence of this phenomenon: 137c strain showed the significant intracellular inhomogeneity of ST occurrence, whereas 4A+ strain hardly did. On the other hand, the SIM observation resolved partially irreversible fine thylakoid transformations caused by the ST-inducing illumination. This fine, irreversible thylakoid transformation was also observed in the STT7 kinase-lacking mutant. This result revealed that the fine thylakoid transformation is not induced solely by the LHCII phosphorylation, suggesting the highly susceptible nature of the thylakoid ultrastructure to the photosynthetic light reactions.

Exciton quenching by oxidized chlorophyll Z across the two adjacent monomers in a photosystem II core dimer.

This study aimed to clarify (1) which pigment in a photosystem II (PSII) core complex is responsible for the 695-nm emission at 77 K and (2) the molecular basis for the oxidation-induced fluorescence quenching in PSII. Picosecond time-resolved fluorescence dynamics was compared between the dimeric and monomeric PSII with and without addition of an oxidant. The results indicated that the excitation-energy flow to the 695-nm-emitting chlorophyll (Chl) at 36 K and 77 K was hindered upon monomerization, clearly demonstrating significant exciton migration from the Chls on one monomer to the 695-nm-emitting pigment on the adjacent monomer. Oxidation of the redox-active Chl, which is named ChlZ caused almost equal quenching of the 684-nm and 695-nm emission bands in the dimer, and lower quenching of the 695-nm band in the monomer. These results suggested two possible scenarios responsible for the 695-nm emission band: (A) Chl11-13 pair and the oxidized ChlZD1 work as the 695-nm emitting Chl and the quenching site, respectively, and (B) Chl29 and the oxidized ChlZD2 work as the 695-nm emitting Chl and the quenching site, respectively.

High-Speed Excitation-Spectral Microscopy Uncovers In Situ Rearrangement of Light-Harvesting Apparatus in Chlamydomonas during State Transitions at Submicron Precision.

Photosynthetic organisms adjust to fluctuating natural light under physiological ambient conditions through flexible light-harvesting ability of light-harvesting complex II (LHCII). A process called state transition is an efficient regulation mechanism to balance the excitations between photosystem II (PSII) and photosystem I (PSI) by shuttling mobile LHCII between them. However, in situ observation of the migration of LHCII in vivo remains limited. In this study, we investigated the in vivo reversible changes in the intracellular distribution of the chlorophyll (Chl) fluorescence during the light-induced state transitions in Chlamydomonas reinhardtii. The newly developed noninvasive excitation-spectral microscope provided powerful spectral information about excitation-energy transfer between Chl-a and Chl-b. The excitation spectra were detected through the fluorescence emission in the 700-750-nm spectral range, where PSII makes the main contribution, though PSI still makes a non-negligible contribution at room temperature. The technique is sensitive to the Chl-b spectral component specifically bound to LHCII. Using a PSI-specific 685-nm component also provided visualization of the local relative concentration of PSI within a chloroplast at room temperature. The decrease in the relative intensity of the Chl-b band in state 2 was more conspicuous in the PSII-rich region than in the PSI-rich region, reflecting the dissociation of LHCII from PSII. We observed intracellular redistributions of the Chl-b-related light-harvesting abilities within a chloroplast during the state transitions. This observation implies the association of the state transitions with the morphological changes in the thylakoid membrane.

Development of a Multicolor Line-Focus Microscope for Rapid Acquisitions of Excitation Spectra.

To conduct rapid microscope observations with the excitation spectral measurement for photosynthetic organisms, a wavelength-dispersive line-focus microscope was developed. In the developed system, fluorescence signals at multiple positions on a sample excited with different wavelengths can be detected as a two-dimensional image on the EMCCD camera at the same time. Using the developed system, one can obtain excitation spectra at every pixel over the excitation wavelength range from 635 to 695 nm, which covers the full range of the Qy bands of both chlorophyll-a and chlorophyll-b. Recording the reference laser spectra at the same time ensures robust measurement against the moderate spectral fluctuation in the excitation laser. Using an objective lens with a numerical aperture of 0.9, the lateral and axial resolutions of 0.56 and 1.08 µm, respectively, were achieved. The theoretically limited and experimentally estimated spectral resolutions of the excitation spectral measurement were 0.86 and 1.3 nm, respectively. The validity of the system was demonstrated by measuring fluorescent beads and single cells of a model alga, Chlamydomonas reinhardtii. Intrachloroplast inhomogeneity in the relative intensity of the chlorophyll-b band could be visualized in Chlamydomonas cells. The inhomogeneity reflects the intrachloroplast variation in the local peripheral antenna size.

Identification of assembly precursors to photosystems emitting fluorescence at 683 nm and 687 nm by cryogenic fluorescence microspectroscopy.

Photosystem I (PSI) and photosystem II (PSII) play key roles in photoinduced electron-transfer reaction in oxygenic photosynthesis. Assemblies of these PSs can be initiated by illumination of the etiolated seedlings (greening). The study aimed to identify specific fluorescence spectral components relevant to PSI and PSII assembly intermediates emerging in greening seedlings of Zea mays, a typical C4 plant. The different PSII contents between the bundle sheath (BS) and mesophyll (M) cells were utilized to spectrally isolate the precursors to PSI and PSII. The greening Zea mays leaf thin sections were observed with the cryogenic microscope combined with a spectrometer. With the aid of the singular-value decomposition analysis, we could identify four independent fluorescent species, SAS677, SAS685, SAS683, and SAS687, named after their fluorescence peak wavelengths. SAS677 and SAS685 are the dominant components after the 30-minute greening, and the distributions of these components showed no clear differences between M and BS cells, indicating immature cell differentiation in this developing stage. On the other hand, the 1-hour greening resulted in reduced distributions of SAS683 in BS cells leading us to assign this species to PSII precursors. The 2-hour greening induced the enrichment of SAS687 in BS cells suggesting its PSI relevance. Similarity in the peak wavelengths of SAS683 and the reported reaction center of PSII implied their connection. SAS687 showed an intense sub-band at around 740 nm, which can be assigned to the emission from the red chlorophylls specific to the mature PSI.

Redox-state dependent blinking of single photosystem I trimers at around liquid-nitrogen temperature.

Efficient light harvesting in a photosynthetic antenna system is disturbed by a ragged and fluctuating energy landscape of the antenna pigments in response to the conformation dynamics of the protein. This situation is especially pronounced in Photosystem I (PSI) containing red shifted chlorophylls (red Chls) with the excitation energy much lower than the primary donor. The present study was conducted to clarify light-harvesting dynamics of PSI isolated from Synechocystis sp. PCC6803 by using single-molecule spectroscopy at liquid­nitrogen temperatures. Fluorescence emission at around 720 nm from the red Chls in single PSI trimers was monitored at 80-100 K. Intermittent variations in the emission intensities, so-called blinking, were frequently observed. Its time scale lay in several tens of seconds. The blinking amplitude depended on the redox state of the phylloquinone (A1). Electrochromic shifts of Chls induced by the negative charge on A1 were calculated based on the X-ray crystallographic structure. A Chl molecule, Chl-A839 (numbering according to PDB 5OY0), bound near A1 was found to have a large electrochromic shift. This Chl has strong exciton coupling with neighboring Chl (A838) whose site energy was predicted to be determined by interaction with an arginine residue (ArgF84) [Adolphs et al., 2010]. A possible scenario of the blinking was proposed. Conformational fluctuations of ArgF84 seesaw the excitation-energy of Chl-A838, which perturbs the branching ratio of excitation-energy between the red Chl and the cationic form of P700 as a quencher. The electrochromic shift of Chl-A839 enhances the effect of the conformation dynamics of ArgF84.

Imaging of intracellular rearrangement of photosynthetic proteins in Chlamydomonas cells upon state transition.

Oxygenic photosynthesis is conducted by two photoactive units, photosystem I (PSI) and photosystem II (PSII), that utilize light energy to generate the electron flow from water to NADPH. Photosynthetic organisms have developed a mechanism called state transition (ST) to regulate the excitation balance between the two units, since the balance is constantly disturbed by fluctuation in light quality. The traditional ST model assumes shuttling of a light-harvesting complex called LHCII between the two PSs. However, there has been no direct observation of the intracellular rearrangements of LHCII upon ST, which is crucial in order to evaluate the validity of the traditional ST model. Here, the intracellular distributions of the two PSs and LHCII are visualized by using a novel cryogenic optical microscope. The calculated Pearson's correlation coefficient between the relative fluorescence intensity of LHCII and the fluorescence intensity ratio of PSII to PSI provided information about the degree of co-localization of these components. The analysis indicated that the relative emission intensity from LHCII is stronger in the PSII-abundant region than in the PSI-abundant one in both states. On the other hand, a statistical analysis by Welch's test indicated that Pearson's correlation coefficient is significantly higher in state1 than state2, probably reflecting the movement of LHCII from PSII to PSI upon state transition. The study also found an independent cell group in which degree of ST was between those observed for fully converted cells. These cells tended to show lower correlation coefficients than the fully converted ones. This was explained by assuming the existence of free LHCII, which moves to but remains unconnected to PSI in state2.

Red shift in the spectrum of a chlorophyll species is essential for the drought-induced dissipation of excess light energy in a poikilohydric moss, Bryum argenteum.

Some mosses are extremely tolerant of drought stress. Their high drought tolerance relies on their ability to effectively dissipate absorbed light energy to heat under dry conditions. The energy dissipation mechanism in a drought-tolerant moss, Bryum argenteum, has been investigated using low-temperature picosecond time-resolved fluorescence spectroscopy. The results are compared between moss thalli samples harvested in Antarctica and in Japan. Both samples show almost the same quenching properties, suggesting an identical drought tolerance mechanism for the same species with two completely different habitats. A global target analysis was applied to a large set of data on the fluorescence-quenching dynamics for the 430-nm (chlorophyll-a selective) and 460-nm (chlorophyll-b and carotenoid selective) excitations in the temperature region from 5 to 77 K. This analysis strongly suggested that the quencher is formed in the major peripheral antenna of photosystem II, whose emission spectrum is significantly broadened and red-shifted in its quenched form. Two emission components at around 717 and 725 nm were assigned to photosystem I (PS I). The former component at around 717 nm is mildly quenched and probably bound to the PS I core complex, while the latter at around 725 nm is probably bound to the light-harvesting complex. The dehydration treatment caused a blue shift of the PS I emission peak via reduction of the exciton energy flow to the pigment responsible for the 725 nm band.

Fluorescence property of photosystem II protein complexes bound to a gold nanoparticle.

Development of an efficient photo-anode system for water oxidation is key to the success of artificial photosynthesis. We previously assembled photosystem II (PSII) proteins, which are an efficient natural photocatalyst for water oxidation, on a gold nanoparticle (GNP) to prepare a PSII-GNP conjugate as an anode system in a light-driven water-splitting nano-device (Noji et al., J. Phys. Chem. Lett., 2011, 2, 2448-2452). In the current study, we characterized the fluorescence property of the PSII-GNP conjugate by static and time-resolved fluorescence measurements, and compared with that of free PSII proteins. It was shown that in a static fluorescence spectrum measured at 77 K, the amplitude of a major peak at 683 nm was significantly reduced and a red shoulder at 693 nm disappeared in PSII-GNP. Time-resolved fluorescence measurements showed that picosecond components at 683 nm decayed faster by factors of 1.4-2.1 in PSII-GNP than in free PSII, explaining the observed quenching of the major fluorescence peak. In addition, a nanosecond-decay component arising from a 'red chlorophyll' at 693 nm was lost in time-resolved fluorescence of PSII-GNP, probably due to a structural perturbation of this chlorophyll by interaction with GNP. Consistently with these fluorescence properties, degradation of PSII during strong-light illumination was two times slower in PSII-GNP than in free PSII. The enhanced durability of PSII is an advantageous property of the PSII-GNP conjugate in the development of an artificial photosynthesis device.

Structure-Based Modeling of Fluorescence Kinetics of Photosystem II: Relation between Its Dimeric Form and Photoregulation.

A photosystem II-enriched membrane (PSII-em) consists of the PSII core complex (PSII-cc) which is surrounded by peripheral antenna complexes. PSII-cc consists of two core antenna (CP43 and CP47) and the reaction center (RC) complex. Time-resolved fluorescence spectra of a PSII-em were measured at 77 K. The data were globally analyzed with a new compartment model, which has a minimum number of compartments and is consistent with the structure of PSII-cc. The reliability of the model was investigated by fitting the data of different experimental conditions. From the analysis, the energy-transfer time constants from the peripheral antenna to CP47 and CP43 were estimated to be 20 and 35 ps, respectively. With an exponential time constant of 320 ps, the excitation energy was estimated to accumulate in the reddest chlorophyll (Red Chl), giving a 692 nm fluorescence peak. The excited state on the Red Chl was confirmed to be quenched upon the addition of an oxidant, as reported previously. The calculations based on the Förster theory predicted that the excitation energy on Chl29 is quenched by ChlZD1(+), which is a redox active but not involved in the electron-transfer chain, located in the D1 subunit of RC, in the other monomer with an exponential time constant of 75 ps. This quenching pathway is consistent with our structure-based simulation of PSII-cc, which assigned Chl29 as the Red Chl. On the other hand, the alternative interpretation assigning Chl26 as the Red Chl was not excluded. The excited Chl26 was predicted to be quenched by another redox active ChlZD2(+) in the D2 subunit of RC in the same monomer unit with an exponential time constant of 88 ps.

Development of a novel cryogenic microscope with numerical aperture of 0.9 and its application to photosynthesis research.

A novel cryogenic optical-microscope system was developed in which the objective lens is set inside of the cryostat adiabatic vacuum space. Being isolated from the sample when it was cooled, the objective lens was maintained at room temperature during the cryogenic measurement. Therefore, the authors were able to use a color-aberration corrected objective lens with a numerical aperture of 0.9. The lens is equipped with an air vent for compatibility to the vacuum. The theoretically expected spatial resolutions of 0.39µm along the lateral direction and 1.3µm along the axial direction were achieved by the developed system. The system was applied to the observations of non-uniform distributions of the photosystems in the cells of a green alga, Chlamydomonas reinhardtii, at 94K. Gaussian decomposition analysis of the fluorescence spectra at all the pixels clearly demonstrated a non-uniform distribution of the two photosystems, as reflected in the variable ratios of the fluorescence intensities assigned to photosystem II and to those assigned to photosystem I. The system was also applied to the fluorescence spectroscopy of single isolated photosystem I complexes at 90K. The fluorescence, assigned to be emitted from a single photosystem I trimer, showed an intermittent fluctuation called blinking, which is typical for a fluorescence signal from a single molecule. The vibronic fluorescence bands at around 790nm were observed for single photosystem I trimers, suggesting that the color aberration is not serious up to the 800nm spectral region.

Scrambled self-assembly of bacteriochlorophylls c and e in aqueous Triton X-100 micelles.

Bacteriochlorophyll (BChl) e was coassembled with BChl c in Triton X-100 micelles in aqueous solutions. The Qy absorption bands of the coaggregates were positioned between those of aggregates consisting solely of BChl c or e. The electronic absorption spectra of the coaggregates could not be reproduced by linear combinations of the spectra of the aggregates consisting solely of each pigment, but they were in line with the simulated spectra for the self-aggregates in which both BChls were randomly distributed. These suggest that BChls c and e are not spatially separated; they are homogenously distributed over the self-aggregates to give electronic spectra that are different from those of the aggregate consisting solely of each pigment. Deaggregation of the scrambled self-aggregates by excess Triton X-100 did not produce any spectral components assigned to an aggregate consisting solely of either BChl c or e. Acid-induced decomposition of the scrambled aggregates showed different kinetics from those of the aggregates consisting solely of each pigment. These also support the homogeneous distribution of BChls c and e in the scrambled self-aggregates. These results will be useful to investigate the major light-harvesting antenna systems of green photosynthetic bacteria that contain two kinds of chlorosomal BChls.

Arabitol provided by lichenous fungi enhances ability to dissipate excess light energy in a symbiotic green alga under desiccation.

Lichens are drought-resistant symbiotic organisms of mycobiont fungi and photobiont green algae or cyanobacteria, and have an efficient mechanism to dissipate excess captured light energy into heat in a picosecond time range to avoid photoinhibition. This mechanism can be assessed as drought-induced non-photochemical quenching (d-NPQ) using time-resolved fluorescence spectroscopy. A green alga Trebouxia sp., which lives within a lichen Ramalina yasudae, is one of the most common green algal photobionts. This alga showed very efficient d-NPQ under desiccation within the lichen thallus, whereas it lost d-NPQ ability when isolated from R. yasudae, indicating the importance of the interaction with the mycobiont for d-NPQ ability. We analyzed the water extracts from lichen thalli that enhanced d-NPQ in Trebouxia. Of several sugar compounds identified in the water extracts by nuclear magnetic resonance (NMR), mass spectrometry (MS) and gas chromatography (GC) analyses, only d-arabitol recovered d-NPQ in isolated Trebouxia to a level similar to that detected for R. yasudae thallus. Other sugar compounds did not help the expression of d-NPQ at the same concentrations. Thus, arabitol is essential for the expression of d-NPQ to dissipate excess captured light energy into heat, protecting the photobiont from photoinhibition. The relationship between mycobionts and photobionts is, therefore, not commensalism, but mutualism with each other, as shown by d-NPQ expression.

Photosystem II does not possess a simple excitation energy funnel: time-resolved fluorescence spectroscopy meets theory.

The experimentally obtained time-resolved fluorescence spectra of photosystem II (PS II) core complexes, purified from a thermophilic cyanobacterium Thermosynechococcus vulcanus, at 5-180 K are compared with simulations. Dynamic localization effects of excitons are treated implicitly by introducing exciton domains of strongly coupled pigments. Exciton relaxations within a domain and exciton transfers between domains are treated on the basis of Redfield theory and generalized Förster theory, respectively. The excitonic couplings between the pigments are calculated by a quantum chemical/electrostatic method (Poisson-TrEsp). Starting with previously published values, a refined set of site energies of the pigments is obtained through optimization cycles of the fits of stationary optical spectra of PS II. Satisfactorily agreement between the experimental and simulated spectra is obtained for the absorption spectrum including its temperature dependence and the linear dichroism spectrum of PS II core complexes (PS II-CC). Furthermore, the refined site energies well reproduce the temperature dependence of the time-resolved fluorescence spectrum of PS II-CC, which is characterized by the emergence of a 695 nm fluorescence peak upon cooling down to 77 K and the decrease of its relative intensity upon further cooling below 77 K. The blue shift of the fluorescence band upon cooling below 77 K is explained by the existence of two red-shifted chlorophyll pools emitting at around 685 and 695 nm. The former pool is assigned to Chl45 or Chl43 in CP43 (Chl numbering according to the nomenclature of Loll et al. Nature2005, 438, 1040) while the latter is assigned to Chl29 in CP47. The 695 nm emitting chlorophyll is suggested to attract excitations from the peripheral light-harvesting complexes and might also be involved in photoprotection.

Study of cell-differentiation and assembly of photosynthetic proteins during greening of etiolated Zea mays leaves using confocal fluorescence microspectroscopy at liquid-nitrogen temperature.

Fluorescence microspectroscopy observations were used to study the processes of cell differentiation and assemblies of photosynthesis proteins in Zea mays leaves under the greening process. The observations were done at 78K by setting the sample in a cryostat to avoid any undesired progress of the greening process during the measurements. The lateral and axial spatial resolutions of the system were 0.64µm and 4.4µm, respectively. The study revealed the spatial distributions of protochlorophyllide (PChld) in both the 632-nm-emitting and 655-nm-emitting forms within etiolated Zea mays leaves. The sizes of the fluorescence spots attributed to the former were larger than those of the latter, validating the assignment of the former and latter to the prothylakoid and prolamellar bodies, respectively. In vivo microspectroscopy observations of mature Zea mays leaves confirmed the different photosystem II (PS I)/photosystem I (PS II) ratio between the bundle sheath (BS) and mesophyll (MS) cells, which is specific for C4-plants. The BS cells in Zea mays leaves 1h after the initiation of the greening process tended to show fluorescence spectra at shorter wavelength side (at around 679nm) than the MS cells (at around 682nm). The 679-nm-emitting chlorophyll-a form observed mainly in the BS cells was attributed to putative precursor complexes to PS I. The BS cells under 3-h greening showed higher relative intensities of the PS I fluorescence band at around 735nm, suggesting the reduced PS II amount in the BS cells in this greening stage.